The tobacco specific nitrosamines N'-nitrosonornicotine (NNN) and 4-0(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) which have been isolated from chewing tobacco (2.4 to 39 Mug/g) and from mainstgream cigarette smoke (0.15 to 6.1 Mug/cigarette) are carcinogenic in strain A mice, F-344 rats and Syrian golden hamsters. Studies of their urinary metabolites suggest that they are activated by Alpha-carbon hydroxylation which yields electrophilic intermediates. After in vitro or in vivo activation radiolabelled NNN or NNK alkylate cellular macromolecules: DNA, RNA and protein. A main objective of this proposal is to develop a specific and sensitive analytical method (e.g. radioimmunoassays) for the detection f O6- and 7-alkylguanine residues isolated from animal and human cells exposed to NNN and NNK. In this project, O6- and 7-alkylguanosine corresponding to some NNN- and NNK-DNA adducts will be synthesized and used as haptens. Specific antibodies will be raised by immunization of rabbits with conjugates of those haptens coupled to keyhold limpet hemocyanin. We will try to correlate levels and persistence of these alkylated deoxyguanosines with the susceptibility of animal tissues to develop tumors when exposed to activated NNN and NNK. The radioimmunoassays developed in this project should enable us to measure the extent of DNA alkylation when epthelial cells of the oral cavity are exposed in vivo to tobacco smoke or chewing tobacco. We will determine if the levels of some tobacco-specific nitrosamine-DNA adducts can be correlated to the high incidences of oral cavity cancer among chewers and smokers, including those who drink.